Assessment of the Effects of Tyrosine Protein Kinase Inhibitors

Anne L. Burkhardt1, Joseph B. Bolen1

1 Bristol‐Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 11.5
DOI:  10.1002/0471142735.im1105s07
Online Posting Date:  May, 2001
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The tyrosine protein kinases are enzymes that are important in cellular signal transduction. Therefore, inhibition of TPKs provides an important means of investigating and potentially controlling many signaling pathways. The first basic protocol in this unit describes an assay of the inhibitory effects of TPK inhibitors in vitro on a specific TPK that has been immune‚Äźprecipitated from cell lysates. An assay of the effects of several TPK inhibitors on TPKs in vivo in activated cells is also provided. Although the example used here is a nonreceptor TPK, these protocols can be used to assay the effects of inhibitors on receptor TPKs as well.

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Table of Contents

  • Basic Protocol 1: Assessing Effects of Inhibitors on TPK Assays Using Cell Lysates
  • Basic Protocol 2: Assessing Tyrosine Protein Kinase Inhibitors Using Intact Cultured T Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
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Basic Protocol 1: Assessing Effects of Inhibitors on TPK Assays Using Cell Lysates

  • Cell culture
  • recipe10 mM inhibitor stock solution: genistein, herbimycin A, peptide A, or tyrophostin (Reagents and Solutions)
  • 1 M MgCl 2 ( appendix 2A)
  • 100 µM ATP solution (unit 11.4)
  • 5 mCi/ml [γ32P]ATP (3000 Ci/mmol; Du Pont NEN)
  • 2× SDS/sample buffer (unit 8.4)
  • Eppendorf model 5432 mixer
  • Gel dryer
  • Additional reagents and equipment for immunoprecipitation of TPKs (unit 11.4), SDS‐PAGE by the Laemmli system (unit 8.4), and autoradiography ( appendix 3A)
CAUTION: Perform all steps involving 32P behind a plexiglass shield, observing appropriate safety regulations. Provide waste containers for both dry and liquid radioactive waste.

Basic Protocol 2: Assessing Tyrosine Protein Kinase Inhibitors Using Intact Cultured T Cells

  • Culture of HPB‐ALL human T cell leukemia cells
  • Appropriate complete culture medium ( appendix 2A)
  • recipe10 mM inhibitor stock solution: e.g., genistein, herbimycin A, or tyrophostin (Reagents and Solutions)
  • Dimethylsulfoxide (DMSO)
  • Agonist antibody: OKT3 ( Ortho)
  • Crosslinking antibody: rabbit anti‐mouse IgG ( Organon Teknika Cappel)
  • 5× lysis buffer with protease inhibitors (unit 11.4)
  • or 5× SDS/sample buffer (unit 8.4)
  • Additional reagents and equipment for SDS‐PAGE using the Laemmli system (unit 8.4), electroblotting (unit 8.10), and antiphosphotyrosine blotting (unit 11.3)
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Literature Cited

   Akiyama, T., Ishida, J., Nakagawa, S., Ogawara, H., Watanabe, S., Itoh, N., Shibuya, M. K., and Fukami, Y. 1987. Genistein, a specific inhibitor of tyrosine‐specific protein kinases. J. Biol. Chem. 262:5592‐5595.
   DeBoer, C., Melman, P.A., Wnuk, R.J., and Peterson, D.H. 1970. Geldanamycin, a new antibiotic. J. Antibiot. (Tokyo) 23:442‐447.
   Fukami, Y., Sato, K., Ikeda, K., Kamisango, K., Koizumi, K., and Matsuno, T. 1993. Evidence for autoinhibitory regulation of the c‐src gene product, possible interaction between the src homology 2 domain and autophosphorylation site. J. Biol. Chem. 268:1132‐1140.
   Gazit, A., Yaish, P., Gilon, C., and Levitzki, A. 1989. Tyrophostins I: Synthesis and biological activity of protein tyrosine kinase inhibitors. J. Med. Chem. 32:2344‐2352.
   Geahlen, R.L. and McLaughlin, J.L. 1989. Piceatannol (3,4,5′‐tetrahydroxy‐trans‐stilbene) is a naturally occurring protein‐tyrosine kinase inhibitor. Biochem. Biophys. Res. Commun. 165:241‐245.
   Geissler, J.F., Traxler, P., Regenass, U., Murray, B.J., and Roesel, J.L. 1990. Thiazole‐diones. Biochemical and biological activity of a novel class of tyrosine protein kinase inhibitors. J. Biol. Chem. 265:22255‐22261.
   Isshiki, K., Naganawa, H., and Takeuchi, T. 1989. Isolation of a tyrosine kinase inhibitor, lavendustin A, from Streptomyces griseolavendus. J. Natl. Prod. 52:1252‐1257.
   Jayasuriya, H., Koonchanok, N.M., Geahlen, R.L., McLaughlin, J.L., and Chang, C.J. 1992. Emodin, a protein tyrosine kinase inhibitor from Polygonium cuspidatum. J. Natl. Prod. 55:696‐698.
   Nakamura, J., Iitaka, Y., Imoto, M., Isshiki, K., Naganawa, H., Takeuchi, T., and Umezawa, H. 1986. The structure of an epidermal growth factor receptor kinase inhibitor, erbstatin. J. Antibiot. (Tokyo) 39:314‐315.
   Sato, K., Miki, S., Tachibana, H., Hayashi, F., Akiyama, T., and Fukami, Y. 1990. A synthetic peptide corresponding to residues 137 to 157 of p60v‐src inhibits tyrosine‐specific protein kinases. Biochem. Biophys. Res. Commun. 171:1152‐1159.
   Shoelson, S.E., White, M.F., and Kahn, C.R. 1989. Nonphosphorylatable substrate analogs selectively block autophosphorylation and activation of the insulin receptor, epidermal growth factor, and pp60v‐src kinases. J. Biol. Chem. 264:7831‐7836.
   Smith, J.A., Colorimetric methods. 1987. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.F. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 10.1.1‐10.1.3. Greene Publishing Associates and John Wiley & Sons, New York.
   Tapley, P., Lamballe, F., and Barbacid, M. 1992. K252a is a selective inhibitor of the tyrosine protein kinase activity of the trk family of oncogenes and neurotrophin receptors. Oncogene 7:371‐381.
   Uehara, Y., Hori, M., Takeuchi, T., and Umezawa, H. 1985. Screening of agents which convert “transformed morphology”: Identification of an active agent as herbimycin and its inhibition of intracellular src kinase. Jpn. J. Cancer Res. 76:672‐675.
   Wong, T.W. and Goldberg, A.R. 1981. Synthetic peptide fragment of src gene product inhibits the src protein kinase and crossreacts immunologically with avian onc kinases and cellular phosphoproteins. Proc. Natl. Acad. Sci. U.S.A. 78:7412‐7416.
Key References
   Burke, T.R. Jr. 1992. Protein‐tyrosine kinase inhibitors. Drugs Future 17:119‐131.
  Provide recent general reviews about TPK inhibitors.
   Workman, P., Brunton, V.G., and Robins, D.J. 1992. Tyrosine kinase inhibitors. Semin. Cancer Biol. 3:369‐381.
  Provide significant detail on use of TPK inhibitors.
   Levitzki, A., Gazit, A., Osherov, N., Posner, I., and Gilon, C. 1991. Inhibition of protein‐tyrosine kinases by tyrophostins. Methods Enzymol. 201:347‐370.
  Describe specific cases where various TPK inhibitors have been used to analyze T cell signal transduction.
   Uehara, Y. and Fukazawa, H. 1991. Use and selectivity of herbimycin A as inhibitor of protein‐tyrosine kinases. Methods Enzymol. 201:370‐385.
   Graber, M., June, C.H., Samelson, L.E., and Weiss, A. 1992. The protein tyrosine kinase inhibitor herbimycin A, but not genistein, specifically inhibits signal transduction by the T cell antigen receptor. Int. Immunol. 4:1201‐1210.
   June, C.H., Fletcher, M.C., Ledbetter, J.A., Schieven, G.L., Siegel, J.N., Phillips, A.F., and Samelson, L.E. 1990. Inhibition of tyrosine phosphorylation prevents T cell receptor‐mediated signal transduction. Proc. Natl. Acad. Sci. U.S.A. 87:7722‐7726.
   Mustelin, T., Coggeshall, K.M., Isakov, N., and Altman, A. 1990. T cell antigen receptor‐mediated activation of phospholipase C requires tyrosine phosphorylation. Science (Wash. DC) 247:1584‐1587.
   Stanley, J.B., Gorczynski, R., Huang, C., Love, J., and Mills, G.B. 1990. Tyrosine phosphorylation is an obligatory event in IL‐2 secretion. J. Immunol. 145:2189‐2198.
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